Bidirectional Solid-State Fermentation of Highland Barley by Edible Fungi to Improve Its Functional Components, Antioxidant Activity and Texture Characteristics.

In food industry, the characteristics of food substrate could be improved through its bidirectional solid-state fermentation (BSF) by fungi, because the functional components were produced during BSF. Six edible fungi were selected for BSF to study their effects on highland barley properties, such as functional components, antioxidant activity, and texture characteristics. After BSF, the triterpenes content in Ganoderma lucidum and Ganoderma leucocontextum samples increased by 76.57 and 205.98%, respectively, and the flavonoids content increased by 62.40% (Phellinus igniarius). Protein content in all tests increased significantly, with a maximal increase of 406.11% (P. igniarius). Proportion of indispensable amino acids increased significantly, with the maximum increase of 28.22%. Lysine content increased largest by 437.34% to 3.310 mg/g (Flammulina velutipes). For antioxidant activity, ABTS radical scavenging activity showed the maximal improvement, with an increase of 1268.95%. Low-field NMR results indicated a changed water status of highland barley after fermentation, which could result in changes in texture characteristics of highland barley. Texture analysis showed that the hardness and chewiness of the fermented product decreased markedly especially in Ganoderma lucidum sample with a decrease of 77.96% and 58.60%, respectively. The decrease indicated a significant improvement in the taste of highland barley. The results showed that BSF is an effective technology to increase the quality of highland barley and provide a new direction for the production of functional foods.

The transcription factor GCN4 contributes to maintaining intracellular amino acid contents under nitrogen-limiting conditions in the mushroom Ganoderma lucidum.

BACKGROUND: Edible mushrooms are delicious in flavour and rich in high-quality protein and amino acids required by humans. A transcription factor, general control nonderepressible 4 (GCN4), can regulate the expression of genes involved in amino acid metabolism in yeast and mammals. A previous study revealed that GCN4 plays a pivotal role in nitrogen utilization and growth in Ganoderma lucidum. However, its regulation is nearly unknown in mushrooms. RESULTS: In this study, we found that the amino acid contents reached 120.51 mg per gram of mycelia in the WT strain under 60 mM asparagine (Asn) conditions, but decreased by 62.96% under 3 mM Asn conditions. Second, silencing of gcn4 resulted in a 54.2% decrease in amino acid contents under 60 mM Asn, especially for the essential and monosodium glutamate-like flavour amino acids. However, these effects were more pronounced under 3 mM Asn. Third, silencing of gcn4 markedly inhibited the expression of amino acid biosynthesis and transport genes. In addition, GCN4 enhanced the tricarboxylic acid cycle (TCA) and glycolytic pathway and inhibited the activity of target of rapamycin complex 1 (TORC1), thus being beneficial for maintaining amino acid homeostasis. CONCLUSION: This study confirmed that GCN4 contributes to maintaining the amino acid contents in mushrooms under low concentrations of nitrogen. In conclusion, our study provides a research basis for GCN4 to regulate amino acid synthesis and improve the nutrient contents of edible mushrooms.

Mitochondrial pyruvate carrier influences ganoderic acid biosynthesis in Ganoderma lucidum.

Mitochondrial pyruvate carriers (MPCs), located in the inner membrane of mitochondria, are essential carriers for pyruvate to enter mitochondria. MPCs regulate a wide range of intracellular metabolic processes, such as glycolysis, the tricarboxylic acid cycle (TCA cycle), fatty acid metabolism, and amino acid metabolism. However, the metabolic regulation of MPCs in macrofungi is poorly studied. We studied the role of MPCs in Ganoderma lucidum (GlMPC) on ganoderic acid (GA) biosynthesis regulation in G. lucidum. In this study, we found that the mitochondrial/cytoplasmic ratio of pyruvate was downregulated about 75% in GlMPC1- and GlMPC2-silenced transformants compared with wild type (WT). In addition, the GA content was 17.72 mg/g and increased by approximately 50% in GlMPC1- and GlMPC2-silenced transformants compared with WT. By assaying the expression levels of three key enzymes and the enzyme activities of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH) of the TCA cycle in GlMPC1- and GlMPC2-silenced transformants, it was found that the decrease in GlMPCs activity did not significantly downregulate the TCA cycle rate, and the enzyme activity of IDH increased by 44% compared with WT. We then verified that fatty acid ß-oxidation (FAO) supplements the TCA cycle by detecting the expression levels of key enzymes involved in FAO. The results showed that compared with WT, the GA content was 1.14 mg/g and reduced by approximately 40% in co-silenced transformants. KEY POINTS: • GlMPCs affects the distribution of pyruvate between mitochondria and the cytoplasm. • Acetyl-CoA produced by FAO maintains the TCA cycle. • Acetyl-CoA produced by FAO promotes the accumulation of GA.

Continuous-Wave Raman Lasing from Metal-Linked Organic Dimer Microcrystals.

Stimulated Raman scattering offers an alternative strategy to explore continuous-wave (c.w.) organic lasers, which, however, still suffers from the limitation of inadequate Raman gain in organic material systems. Here we propose a metal-linking approach to enhance the Raman gain of organic molecules. Self-assembled microcrystals of the metal linked organic dimers exhibit large Raman gain, therefore allowing for c.w. Raman lasing. Furthermore, broadband tunable Raman lasing is achieved in the organic dimer microcrystals by adjusting excitation wavelengths. This work advances the understanding of Raman gain in organic molecules, paving a way for the design of c.w. organic lasers.

Phospholipase D and phosphatidic acid mediate regulation in the biosynthesis of spermidine and ganoderic acids by activating GlMyb in Ganoderma lucidum under heat stress.

Polyamines are essential for all kinds of organisms and take part in the regulation of multiple biological processes. Our previous study revealed that heat stress promoted the conversion of putrescine to spermidine and subsequently promoted the accumulation of ganoderic acids (GAs). However, how heat stress affects polyamine homeostasis remains unclear. To explore the underlying mechanism by which heat stress promoted spermidine biosynthesis, we assessed the effects of signalling molecules that respond to heat stress on spermidine biosynthesis. Our data suggested that heat stress-induced spermidine biosynthesis and GAs accumulation via a phospholipase D (PLD)-mediated phosphatidic acid (PA) signal. Further research revealed that the transcription factor GlMyb promoted spermidine biosynthesis via regulating spermidine synthase genes (spds1 and spds2) expression by directly bonding to their promoters and it responded to heat stress and PA signal. In summary, heat stress activated GlMyb by PLD-mediated PA signalling and subsequently induced the expression of spds1 and spds2 to promote the biosynthesis of spermidine and the accumulation of GAs. Our findings firstly reveal a detailed mechanism by which heat signalling regulates polyamine homeostasis by PLD-mediated PA signal in fungi and provide a greater understanding of how organisms alter polyamine levels in response to environmental changes.

GCN4 Enhances the Transcriptional Regulation of AreA by Interacting with SKO1 To Mediate Nitrogen Utilization in Ganoderma lucidum.

Fungi utilize a wide range of nitrogen to adapt their metabolism. The transcription factor GCN4 has a pivotal role in nitrogen metabolism. However, the mechanism by which GCN4 regulates nitrogen utilization in Ganoderma lucidum is not well understood. In this study, we found that GCN4 physically interacts with SKO1, a bZIP (basic leucine zipper) transcription factor. GCN4 cooperated with SKO1 to positively regulate nitrogen utilization, especially for the expression of areA. Electrophoretic mobility shift assays (EMSA) indicate that GCN4 directly binds to the areA promoter region. Further affinity analysis through biolayer interferometry (BLI) experiments and surface plasmon resonance (SPR) confirmed that GCN4 specifically binds to the promoter region of areA with a strong binding affinity to activate the transcription of areA. In contrast, SKO1 showed no specified binding effect on the areA promoter. However, SKO1 activates the expression of the areA by forming a complex with GCN4, which exhibits a 14.2-fold-higher affinity than GCN4 alone. Furthermore, the presence of SKO1 promotes the stability of GCN4 protein. Accordingly, our study found that the transcription factor SKO1 enhances the transcriptional activity of GCN4 on its target gene areA by interacting with GCN4. Our study illustrates a specific regulatory mechanism for the involvement of GCN4 and SKO1 in nitrogen utilization, which provides innovative insight into the regulation of nitrogen utilization in fungi. IMPORTANCE Nitrogen is an essential nutrient for cell growth and proliferation. Limitations of nitrogen availability in organisms elicit a series of rapid transcriptional reprogramming mechanisms, which involve the participation of many transcription factors. However, the specific mechanism of coordination between different transcription factors regulating nitrogen metabolism has not been explored. Our study revealed that GCN4 interacts with SKO1 and that they are both involved in regulating nitrogen utilization by affecting the transcription level of areA. We also found that GCN4 activates transcription by directly binding to the promoter recognition region of areA. SKO1 facilitates the transcription of areA by GCN4 by forming a more stable complex with GCN4. Our study deepens our understanding of the regulatory network of nitrogen metabolism and demonstrates a further level of regulation for transcription factors.

Spermidine Regulates Mitochondrial Function by Enhancing eIF5A Hypusination and Contributes to Reactive Oxygen Species Production and Ganoderic Acid Biosynthesis in Ganoderma lucidum.

Spermidine, a kind of polycation and one important member of the polyamine family, is essential for survival in many kinds of organisms and participates in the regulation of cell growth and metabolism. To explore the mechanism by which spermidine regulates ganoderic acid (GA) biosynthesis in Ganoderma lucidum, the effects of spermidine on GA and reactive oxygen species (ROS) contents were examined. Our data suggested that spermidine promoted the production of mitochondrial ROS and positively regulated GA biosynthesis. Further research revealed that spermidine promoted the translation of mitochondrial complexes I and II and subsequently influenced their activity. With a reduction in eukaryotic translation initiation factor 5A (eIF5A) hypusination by over 50% in spermidine synthase gene (spds) knockdown strains, the activities of mitochondrial complexes I and II were reduced by nearly 60% and 80%, respectively, and the protein contents were reduced by over 50%, suggesting that the effect of spermidine on mitochondrial complexes I and II was mediated through its influence on eIF5A hypusination. Furthermore, after knocking down eIF5A, the deoxyhypusine synthase gene (dhs), and the deoxyhypusine hydroxylase gene (dohh), the mitochondrial ROS level was reduced by nearly 50%, and the GA content was reduced by over 40%, suggesting that eIF5A hypusination contributed to mitochondrial ROS production and GA biosynthesis. In summary, spermidine maintains mitochondrial ROS homeostasis by regulating the translation and subsequent activity of complexes I and II via eIF5A hypusination and promotes GA biosynthesis via mitochondrial ROS signaling. The present findings provide new insight into the spermidine-mediated biosynthesis of secondary metabolites. IMPORTANCE Spermidine is necessary for organism survival and is involved in the regulation of various biological processes. However, the specific mechanisms underlying the various physiological functions of spermidine are poorly understood, especially in microorganisms. In this study, we found that spermidine hypusinates eIF5A to promote the production of mitochondrial ROS and subsequently regulate secondary metabolism in microorganisms. Our study provides a better understanding of the mechanism by which spermidine regulates mitochondrial function and provides new insight into the spermidine-mediated biosynthesis of secondary metabolites.

Trehalose-6-phosphate synthase influences polysaccharide synthesis and cell wall components in Ganoderma lucidum.

Trehalose-6-phosphate synthase (TPS) is a key enzyme that participates in trehalose metabolism, which can synthesize trehalose in a two-step pathway with trehalose phosphatase, but its role in fungi is rarely studied, especially in large basidiomycetes. In this study, the tps gene of Ganoderma lucidum was cloned and named as gltps. And gltps-silenced strains were constructed by RNA interference. In this study, it is found that the extracellular polysaccharide content increased 1.6-2-fold, but there was no significant change on intracellular polysaccharide content in gltps-silenced strains compared with the wild-type (WT) strain. Furthermore, the cell wall compositions of the gltps-silenced strains were also altered, which showed that the chitin and ß-1,3-glucan contents were significantly decreased. Compared with WT, the concentration of chitin decreased by 20%-50% and that of ß-1, 3-glucan decreased by 15%-30%. The study found that the cells of gltps-silenced strains were more sensitive to cell wall stress, which might be due to changes in the compounds and structure of the cell wall. These results showed that gltps had an important effect on carbohydrate metabolism of G. lucidum cells.

Cloning and characterization of phosphoglucose isomerase in Lentinula edodes.

Phosphoglucose isomerase (PGI) is a key enzyme that participates in polysaccharide synthesis, which is responsible for the interconversion of glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P), but there is little research focusing on its role in fungi, especially in higher basidiomycetes. The pgi gene was cloned from Lentinula edodes and named lepgi. Then, the lepgi-silenced strains were constructed by RNA interference. In this study, we found that lepgi-silenced strains had significantly less biomass than the wild-type (WT) strain. Furthermore, the extracellular polysaccharide (EPS) and intracellular polysaccharide (IPS) levels increased 1.5- to 3-fold and 1.5-fold, respectively, in lepgi-silenced strains. Moreover, the cell wall integrity in the silenced strains was also altered, which might be due to changes in the compounds and structure of the cell wall. The results showed that compared to WT, silencing lepgi led to a significant decrease of approximately 40% in the ß-1,3-glucan content, and there was a significant increase of 2-3-fold in the chitin content. These findings provide support for studying the biological functions of lepgi in L. edodes.

Heme Oxygenase/Carbon Monoxide Participates in the Regulation of Ganoderma lucidum Heat-Stress Response, Ganoderic Acid Biosynthesis, and Cell-Wall Integrity.

Carbon monoxide (CO), a product of organic oxidation processes, arises in vivo principally from the enzymatic reaction of heme oxygenase (HO, transcription gene named HMX1). HO/CO has been found to exert many salutary effects in multiple biological processes, including the stress response. However, whether HO/CO is involved in the regulation of the heat-stress (HS) response of Ganoderma lucidum (G. lucidum) is still poorly understood. In this paper, we reported that under heat stress, the HMX1 transcription level, HO enzyme activity, and CO content increased by 5.2-fold, 6.5-fold and 2-fold, respectively. HMX1 silenced strains showed a 12% increase in ganoderic acid (GA) content under HS as analyzed by HPLC. Furthermore, according to Western blot analysis of the protein phosphorylation levels, HMX1 attenuated the increase in phosphorylation levels of slt2, but the phosphorylation levels were prolonged over a 3 h HS time period. The chitin and glucan content in HMX1 silenced strains increased by 108% and 75%, respectively. In summary, these findings showed that the HO/CO system responds to heat stress and then regulates the HS-induced GA biosynthesis and the cell-wall integrity mediated by the Slt-MAPK phosphorylation level in G. lucidum.

Improvement of laccase activity by silencing PacC in Ganoderma lucidum.

Ganoderma lucidum is a representative white-rot fungus that has great potential to degrade lignocellulose biomass. Laccase is recognized as a class of the most important lignin-degrading enzymes in G. lucidum. However, the comprehensive regulatory mechanisms of laccase are still lacking. Based on the genome sequence of G. lucidum, 15 laccase genes were identified and their encoding proteins were analyzed in this study. All of the laccase proteins are predicted to be multicopper oxidases with conserved copper-binding domains. Most laccase proteins were secreted enzymes in addition to Lac14 in which the signal peptide could not be predicted. The activity of all laccases showed the highest level at pH 3.0 or pH 7.0, with total laccase activity of approximately 200 U/mg protein. Silencing PacC resulted in a 5.2 fold increase in laccase activity compared with WT. Five laccase genes (lac1, lac6, lac9, lac10 and lac14) showed an increased transcription levels (approximately 1.5-5.6 fold) in the PacC-silenced strains versus that in WT, while other laccase genes were downregulated or unchanged. The extracellular pH value was about 3.1, which was more acidic in the PacC-silenced strains than in the WT (pH 3.5). Moreover, maintaining the fermentation pH resulted in a downregulation of laccase activity which is induced by silencing PacC. Our findings indicate that in addition to its function in acidification of environmental pH, PacC plays an important role in regulating laccase activity in fungi.

Regulation of glutamine synthetase activity by transcriptional and posttranslational modifications negatively influences ganoderic acid biosynthesis in Ganoderma lucidum.

Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs-silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately five fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one-hybrid assay showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN-1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO-mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi.

Nitric oxide regulates ganoderic acid biosynthesis by the S-nitrosylation of aconitase under heat stress in Ganoderma lucidum.

Nitric oxide (NO) is an important signalling molecule in stress response of organisms. We previously reported that NO decreases heat stress (HS)-induced ganoderic acid (GA) accumulation in Ganoderma lucidum. To explore the mechanisms by which NO modulates GA biosynthesis under HS, the effect of NO on the reactive oxygen species (ROS) content was examined. The results showed that NO decreased the production of mitochondrial ROS (mitROS) by 60% under HS. Further research revealed that NO reduced the mitROS content by inhibiting aconitase (Acon) activity. The GA content in Acon-silenced (Aconi) strains treated with NO donor did not differ significantly from that in untreated Aconi strains. To study the mechanism by which Acon activity is inhibited, the S-nitrosylation level of Acon was determined. Biotin-switch technology and mass spectrometry analysis were used to show that Acon is S-nitrosylated at the Cys-594 amino acid residue. Substitution of Cys-594 with a Ser, which cannot be S-nitrosylated, abolished the responsiveness of Acon to the NO-induced reduction in its enzymatic activity. These findings demonstrate that NO inhibits Acon activity through S-nitrosylation at Cys-594. In summary, these findings describe mechanism by which NO regulates GA biosynthesis via S-nitrosylation of Acon under HS condition in G. lucidum.

Swi6B, an alternative splicing isoform of Swi6, mediates the cell wall integrity of Ganoderma lucidum.

The cell wall integrity (CWI) signaling activates the transcription factor Swi6 through a MAPK signaling cascade in response to cell wall stresses. In this study, we observed two different mRNA variants of swi6 (GlSwi6A and GlSwi6B) existed, due to alternative splicing. Besides, the expression level of GlSwi6B was higher than that of the GlSwi6A mRNA variant. The co-silencing of GlSwi6A and GlSwi6B was more sensitive to cell wall stress compared with WT, resulting in a decrease of 78% and 76% in chitin and ß-1,3-d-glucan content respectively. However, only the overexpression of GlSwi6B decreased the sensitivity to cell wall stress and increased the content of chitin and ß-1,3-d-glucan compared with the WT strain. Furthermore, Y1H, EMSA and BLI assays revealed that the GlSwi6B could bind to the promoters of chitin and glucan synthesis genes (GL24454 and GL18134). However, the binding phenome has not been observed in the isoform GlSwi6A. Taken together, our results found two different transcripts generated from Swi6, in which the alternative splice isoform of GlSwi6B participates in regulating the CWI of G. lucidum. This study provides the first insight into the alternative splicing isoform of GlSwi6B in the regulation of CWI signaling in fungi.

GCN4 Regulates Secondary Metabolism through Activation of Antioxidant Gene Expression under Nitrogen Limitation Conditions in Ganoderma lucidum.

Nitrogen limitation has been widely reported to affect the growth and development of fungi, and the transcription factor GCN4 (general control nonderepressible 4) is involved in nitrogen restriction. Here, we found that nitrogen limitation highly induced the expression of GCN4 and promoted the synthesis of ganoderic acid (GA), an important secondary metabolite in Ganoderma lucidum. The activated GCN4 is involved in regulating GA biosynthesis. In addition, the accumulation of reactive oxygen species (ROS) also affects the synthesis of GA under nitrogen restrictions. The silencing of the gcn4 gene led to further accumulation of ROS and increased the content of GA. Further studies found that GCN4 activated the transcription of antioxidant enzyme biosynthesis genes gr, gst2, and cat3 (encoding glutathione reductase, glutathione S-transferase, and catalase, respectively) through direct binding to the promoter of these genes to reduce the ROS accumulation. In conclusion, our study found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. This provides an essential insight into the understanding of GCN4 transcriptional regulation of the ROS signaling pathway and enriches the knowledge of nitrogen regulation mechanisms in fungal secondary metabolism of G. lucidum.IMPORTANCE Nitrogen has been widely reported to regulate secondary metabolism in fungi. Our study assessed the specific nitrogen regulatory mechanisms in Ganoderma lucidum. We found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. Our research highlights a novel insight that GCN4, the nitrogen utilization regulator, participates in secondary metabolism through ROS signal regulation. In addition, this also provides a theoretical foundation for exploring the regulation of other physiological processes by GCN4 through ROS in fungi.

Controlled Shape Evolution of Pure-MOF 1D Microcrystals towards Efficient Waveguide and Laser Applications.

MOF-based one-dimensional materials have received increasing attention in the nanophotonics field, but it is still difficult in the flexible shape evolution of MOF micro/nanocrystals for desired optical functionalities due to the susceptible solvothermal growth process. Herein, we report on the well-controlled shape evolution of pure-MOF microcrystals with optical waveguide and lasing performances based on a bottom-up and top-down synergistic method. The MOF microcrystals from solvothermal synthesis (bottom-up) enable the evolution from microrods via microtubes to nanowires through a chelating agent-assisted etching process (top-down). The three types of MOF 1D-microstructures with high crystallinity and smooth surfaces all exhibit efficient optical waveguide performance. Furthermore, MOF nanowire with lowest propagation loss served as low-threshold pure-MOF nanolasers with Fabry-Pérot resonance. These results advance the fundamental understanding on the controlled MOF evolution mechanism, and offer a valuable route for the development of pure-MOF-based photonic components with desired functionalities.

Heat stress promotes the conversion of putrescine to spermidine and plays an important role in regulating ganoderic acid biosynthesis in Ganoderma lucidum.

Heat stress (HS) is inescapable environmental stress that can induce the production of ganoderic acids (GAs) in Ganoderma lucidum. Our previous studies found that putrescine (Put) played an inhibitory role in GAs biosynthesis, which appeared to be inconsistent with the upregulated transcription of the Put biosynthetic gene GlOdc under HS. To uncover the mechanism underlying this phenomenon, two spermidine (Spd) biosynthetic genes, GlSpds1 and GlSpds2, were identified and upregulated under HS. Put and Spd increased by 94% and 160% under HS, respectively, suggesting that HS induces polyamine biosynthesis and promotes the conversion of Put to Spd. By using GlSpds knockdown mutants, it is confirmed that Spd played a stimulatory role in GAs biosynthesis. In GlOdc-kd mutants, Put decreased by 62-67%, Spd decreased by approximately 34%, and GAs increased by 15-22% but sharply increased by 75-89% after supplementation with Spd. In GlSpds-kd mutants, Put increased by 31-41%, Spd decreased by approximately 63%, and GAs decreased by 24-32% and were restored to slightly higher levels than a wild type after supplementation with Spd. This result suggested that Spd, rather than Put, is a crucial factor that leads to the accumulation of GAs under HS. Spd plays a more predominant and stimulative role than Put under HS, possibly because the absolute content of Spd is 10 times greater than that of Put. GABA and H2O2, two major catabolites of Spd, had little effect on GAs biosynthesis. This study provides new insight into the mechanism by which environmental stimuli regulate secondary metabolism via polyamines in fungi. KEY POINTS: • HS induces polyamine biosynthesis and promotes the conversion of Put to Spd in G. lucidum. • Put and Spd played the inhibitory and stimulatory roles in regulating GAs biosynthesis, respectively. • The stimulatory role of Spd was more predominant than the inhibitory role of Put in GAs biosynthesis.

Effect of the heme oxygenase gene on mycelial growth and polysaccharide synthesis in Ganoderma lucidum.

The heme oxygenase gene has antioxidant and cytoprotective effects in organisms, but no related research has been conducted in Ganoderma lucidum. For the first time, we cloned the HMX1 gene in G. lucidum. The CDS is 1092 bp in length and encodes 363 amino acids. The HMX1 protein was prokaryotically expressed and purified, and the enzyme activity of the purified protein was measured. The value of Km was 0.699 µM, and Vm was 81.9 nmol BV h-1 nmol-1 protein. By constructing the silencing vector pAN7-dual-HMX1i, the transformants HMX1i1 and HMX1i2 were obtained. Compared with the wild-type (WT), the average growth rate of HMX1i1 and HMX1i2 decreased by 31% and 23%, respectively, and the mycelium biomass decreased by 53% and 48%, respectively. Compared with the WT, the extracellular polysaccharide content of HMX1i1 and HMX1i2 increased by 59% and 51%, and the intracellular polysaccharide content increased by 24% and 22%, respectively. These results indicate that the HMX1 gene affects mycelial growth and polysaccharide synthesis in G. lucidum.

Pure Metal-Organic Framework Microlasers with Controlled Cavity Shapes.

Metal-organic frameworks (MOFs) are an emerging kind of laser material, yet they remain a challenge in the controlled fabrication of crystal nanostructures with desired morphology for tuning their optical microcavities. Herein, the shape-engineering of pure MOF microlasers was demonstrated based on the coordination-mode-tailored method. The one-dimensional (1D) microwires and 2D microplates were selectively fabricated through changing the HCl concentration to tailor the coordination modes. Both the single-crystalline microwires and microplates with strong optical confinement functioned as low-threshold MOF microlasers. Moreover, distinct lasing behaviors of 1D and 2D MOF microcrystals confirm a typical shape-dependent microcavity effect: 1D microwires serve as Fabry-Pérot (FP) resonators, and 2D microplates lead to the whispering-gallery-mode (WGM) microcavities. These results provide a special pathway for the exploitation of MOF-based micro/nanolasers with on-demand functions.

In Ganoderma lucidum, Glsnf1 regulates cellulose degradation by inhibiting GlCreA during the utilization of cellulose.

Cellulose is a by-product of agricultural production and an abundant waste. As a carbon source, cellulose can be degraded and utilized by fungi. Carbon sources, which act as nutrients, not only provide energy but also serve as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Nutrient-sensing pathways prioritize the use of preferred carbon sources and regulate the production of cellulose-degrading enzymes when necessary. Understanding the regulation of the fungal cellulolytic response will become increasingly important because we strive to increase the efficiency of the utilization of these renewable energy sources. Here, we show that Glsnf1, a sucrose-nonfermenting serine-threonine-protein kinase 1 (Snf1)/AMP-activated protein kinase homologue in medicinal macro basidiomycete Ganoderma lucidum, actively responds to carbon alterations and positively regulates cellulase activity and cellulase-related gene transcription. The carbon catabolite repressor CreA, a zinc binuclear cluster transcription factor that mediates the sensing of nutrients and suppression of the transcription of a number of genes necessary for the consumption of a less preferred carbon source, participates in the Glsnf1-mediated regulation of cellulases. Glsnf1 not only negatively regulates the transcription level of the CreA gene but also hinders its localization in the nucleus. Overall, our findings reveal a key nutrient-sensing mechanism that is critical for the modulation of carbon source adaptation in G. lucidum.